Mechanisms that suppress the expression of specific cellular genes, viruses or mobile genetic elements (such as transposons and retroelements) are critical for normal cellular function in a variety of eukaryotes. A number of related processes, discovered independently in plants (Matzke et al., Curr. Opin. Genet. Dev. 11:221-227, 2001), animals (Fire et al., Nature, 391:806-811, 1998) and fungi (Cogoni, Annu. Rev. Microbiol. 55:381-406, 2001), result in the RNA-directed inhibition of gene expression (also known as RNA silencing). Each of these processes is triggered by molecules containing double-stranded RNA (dsRNA) structure, such as transcripts containing inverted repeats or double-stranded RNA intermediates formed during RNA virus replication. Non-dsRNAs, also referred to as aberrant RNAs, may also function as initiators of RNA silencing. Such aberrant RNAs may be converted into dsRNAs by silencing-associated RNA-dependent RNA polymerases (RDRs), which have been identified in plants, fungi and C. elegans (Tuschl, ChemBiochem, 2:239-245, 2001).
Two major classes of small RNAs have been characterized: short interfering RNAs (siRNAs) and microRNAs (miRNAs). The primary transcripts that eventually form miRNAs are transcribed from non-protein-coding miRNA genes. These transcripts form hairpin structures that are then processed by Dicer (or by Dicer-like activities in plants) to yield small RNA duplexes containing 2-base overhangs at each 3′ end. The mature single-stranded miRNA approximately 20-22 nucleotides in length forms by dissociation of the two strands in the duplex, and is selectively incorporated into the RNA-Induced Silencing Complex, or RISC (Zamore, Science, 296:1265-1269, 2002; Tang et al., Genes Dev., 17:49-63, 2003; Xie et al., Curr. Biol. 13:784-789, 2003).
siRNAs are similar in chemical structure to miRNAs, however siRNAs are generated by the cleavage of relatively long double-stranded RNA molecules by Dicer or DCL enzymes (Zamore, Science, 296:1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001). In animals and plants, siRNAs are assembled into RISC and guide the sequence specific ribonucleolytic activity of RISC, thereby resulting in the cleavage of mRNAs, viral RNAs or other RNA target molecules in the cytoplasm. In the nucleus, siRNAs also guide heterochromatin-associated histone and DNA methylation, resulting in transcriptional silencing of individual genes or large chromatin domains.
MicroRNAs in plants and animals function as posttranscriptional regulators of genes involved in a wide range of cellular processes (Bartel, Cell 116:281-297, 2004; He & Hannon, Nat Rev Genet 5:522-531, 2004). In the plant Arabidopsis thaliana, miRNAs regulate mRNAs encoding at least twelve families of transcription factors, several miRNA metabolic factors, and proteins involved in stress responses, metabolism, and hormone signaling (Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004; Kasschau et al., Dev Cell 4:205-217, 2003; Llave et al., Science 297:2053-2056, 2002b; Vazquez et al., Curr Biol 14:346-351, 2004a; Xie et al., Curr Biol 13:784-789, 2003). Plant miRNAs target a disproportionately high number of genes with functions in developmental processes, including developmental timing, control of cell proliferation, meristem cell function, and patterning. Global disruption of miRNA biogenesis or function, or specific disruption of miRNA-target interactions, can result in severe developmental abnormalities (Achard et al., Development 131:3357-3365, 2004; Chen, Science 303:2022-2025, 2004; Emery et al., Curr Biol 13:1768-1774, 2003; Juarez et al., Nature 428:84-88, 2004; Kidner & Martienssen, Nature 428:81-84, 2004; Laufs et al., Development 131:4311-4322, 2004; Mallory et al., Curr Biol 14:1035-1046, 2004; Palatnik et al., Nature 425:257-263, 2003; Tang et al., Genes & Dev 17:49-63 2003; Vaucheret et al., Genes Dev 18:1187-1197, 2004), indicating that miRNA-based regulation is crucial for normal growth and development. This idea is reinforced by the conservation of most miRNAs and their corresponding targets through significant evolutionary time (Bartel, Cell 116:281-297, 2004). MicroRNAs have been identified by direct cloning methods and computational prediction strategies (Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004; Llave et al., Plant Cell 14:1605-1619, 2000a; Park et al., Curr Biol 12:1484-1495, 2002; Reinhart et al., Genes Dev 16:1616-1626, 2002; Sunkar & Zhu, Plant Cell 16:2001-2019, 2004).
Plant miRNAs usually contain near-perfect complementarity with target sites, which are found most commonly in protein-coding regions of the genome. As a result, most (but not all) plant miRNAs function to guide cleavage of targets through a mechanism similar to the siRNA-guided mechanism associated with RNAi (Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004; Kasschau et al., Dev Cell 4:205-217, 2003; Llave et al., Science 297:2053-2056, 2002; Tang et al., Genes & Dev 17:49-63 2003). In contrast, animal miRNAs contain relatively low levels of complementarity to their target sites, which are most commonly found in multiple copies within 3′ untranslated regions of the target transcript (Lewis et al., Cell 115:787-798, 2003; Rajewsky & Socci, Dev Biol 267:529-535, 2004; Stark et al., PLoS Biol 1:E60, 2003). Most animal miRNAs do not guide cleavage, but rather function to repress expression at the translational or co-translational level (Ambros, Cell 113:673-676, 2003; He & Hannon, Nat Rev Genet 5:522-531, 2004). At least some plant miRNAs may also function as translational repressors (Aukerman & Sakai, Plant Cell 15:2730-2741, 2003; Chen, Science 303:2022-2025, 2004). Translation repression is not an inherent activity of animal miRNAs, as miRNAs will guide cleavage if presented with a target containing high levels of complementarity (Doench et al., Genes Dev 17:438-442, 2003; Hutvagner & Zamore, Science 297:2056-2060, 2002; Yekta et al., Science 304:594-596, 2004; Zeng et al., Proc Natl Acad Sci USA 100:9779-9784, 2003).
MicroRNAs form through nucleolytic maturation of genetically defined RNA precursors that adopt imperfect, self-complementary foldback structures. Processing yields a duplex intermediate (miRNA/miRNA*) that ultimately provides the miRNA strand to the effector complex, termed RISC (Khvorova et al., Cell 115:209-216, 2003; Schwarz et al., Cell 115:199-208, 2003). Plants contain four DICER-LIKE (DCL) proteins, one of which (DCL1) is necessary for maturation of most or all miRNA precursors (Kurihara & Watanabe, Proc Natl Acad Sci USA 101:12753-12758, 2004; Park et al., Curr Biol 12:1484-1495, 2002; Reinhart et al., Genes Dev 16:1616-1626, 2002; Schauer et al., Trends Plant Sci 7:487-491, 2002). The DCL1 protein contains an RNA helicase and two RNaseIII-like domains, a central PAZ domain and C-terminal dsRNA binding motifs. Animal miRNA precursor processing requires Drosha, another RNaseIII domain protein, and Dicer in sequential nucleolytic steps (Lee et al., Nature 425:415-419, 2003). HEN1 participates in miRNA biogenesis or stability in plants via a 3′ methylase activity (Boutet et al., Curr Biol 13:843-848, 2003; Park et al., Curr Biol 12:1484-1495, 2002). The dsRNA-binding HYL1 protein is necessary for miRNA biogenesis in cooperation with DCL1 and HEN1 in the nucleus. Based on sequence similarity, HYL1 has been suggested to function like animal R2D2, which is required post-processing during RISC assembly (Han et al., Proc Natl Acad Sci USA 101:1093-1098, 2004; Liu et al., Science 301:1921-1925, 2003; Pham et al., Cell 117:83-94, 2004; Tomari et al., Science 306:1377-1380, 2004; Vazquez et al., Curr Biol 14:346-351, 2004a). In animals, Exportin-5 (Exp5) regulates the transport of pre-miRNAs from the nucleus to the cytoplasm by a Ran-GTP-dependent mechanism (Bohnsack et al., RNA 10:185-191, 2004; Lund et al., Science 303:95-98, 2003; Yi et al., Genes Dev 17:3011-3016, 2003). In Arabidopsis, HST may provide a related function to transport miRNA intermediates to the cytoplasm (Bollman et al., Development 130:1493-1504, 2003). Active miRNA-containing RISC complexes in plants almost certainly contain one or more ARGONAUTE proteins, such as AGO1 (Fagard et al., Proc Natl Acad Sci USA 97:11650-11654, 2000; Vaucheret et al., Genes Dev 18:1187-1197, 2004). Argonaute proteins in animals were shown recently to provide the catalytic activity for target cleavage (Liu et al., Science 305:1437-1441, 2004; Meister et al., Mol Cell 15:185-197, 2004).
In addition to miRNAs, plants also produce diverse sets of endogenous 21-25 nucleotide small RNAs. Most of these differ from miRNAs in that they arise from double-stranded RNA (rather than imperfect foldback structures), in some cases generated by the activity of RNA-DEPENDENT RNA POLYMERASEs (RDRs). Arabidopsis DCL2, DCL3, DCL4, RDR1, RDR2 and RDR6 have known roles in siRNA biogenesis (Dalmay et al., Cell 101:543-553, 2000; Mourrain et al., Cell 101:533-542, 2000; Peragine et al., Genes & Dev 18:2369-2379, 2004; Vazquez et al., Mol Cell 16:69-79, 2004b; Xie et al., PLoS Biol 2:642-652, 2004; Yu et al., Mol Plant Microbe Interact 16:206-216, 2003). For example, DCL3 and RDR2 cooperate in the heterochromatin-associated RNAi pathway, resulting in ˜24-nucleotide siRNAs from various retroelements and transposons, 5S rDNA loci, endogenous direct and inverted repeats, and transgenes containing direct repeats (Xie et al., PLoS Biol 2:642-652, 2004; Zilberman et al., Science 299:716-719, 2003). RDR6 functions in posttranscriptional RNAi of sense transgenes, some viruses, and specific endogenous mRNAs that are targeted by trans-acting siRNAs (ta-siRNAs) (Dalmay et al., Cell 101:543-553, 2000; Mourrain et al., Cell 101:533-542, 2000; Peragine et al., Genes & Dev 18:2369-2379, 2004; Vazquez et al., Mol Cell 16:69-79, 2004b; Yu et al., Mol Plant Microbe Interact 16:206-216, 2003). Ta-siRNAs arise from transcripts that are recognized by RDR6, in cooperation with SGS3, as a substrate to form dsRNA. The dsRNA is processed accurately in 21-nucleotide steps by DCL1 to yield a set of “phased” ta-siRNAs. These ta-siRNAs interact with target mRNAs to guide cleavage by the same mechanism as do plant miRNAs (Peragine et al., Genes & Dev 18:2369-2379, 2004; Vazquez et al., Mol Cell 16:69-79, 2004).
There is a need to develop methods and constructs that can be used to induce targeted RNAi in vivo. It is to such methods and constructs, and related compositions, that this disclosure is drawn.